The Bactericidal Activity of Protein Extracts from Loranthus europaeus Berries: A Natural Resource of
2. Results
2.1. Samples Collection
In Italy,Loranthus europaeusis prevalently diffused in oak forests of Apennines, extending from Central to South Italy. For this study, the forest of Carpanzano (Calabria), located at an altitude of 610 m, was selected. Carpanzano is a continental territory of Calabria, far from the sea, with cold
winters and higher precipitations during spring and fall. Visible tufts ofL. europaeuswere scattered on numerousQ. pubescenstrees and samples were collected during winter, when mistletoe twigs are leafless, and fruits acquire a bright yellow color (Figure1).
Figure 1.(A,B) Ripen berries ofL. europaeus; (C) oak tree hostingL. europaeustwigs; (D) the sampling site (Carpanzano forest, Calabria, Italy).
2.2. Preparation of Protein Extracts fromLoranthus europaeusBerries and Plant Extracts Yield
With the aim to recover and isolate new plant protein agents with antimicrobial activity, that can possibly be used as natural preservatives in the food and pharmaceutical industries, an efficient protein extraction method fromLoranthus europaeusberries was developed. Indeed, the extraction efficiency is strongly affected by several factors such as the starting plant material, the buffer composition and the method used as well as the presence of interfering substances [24]. It is worth noting that at present there are relatively few reports on the extraction protocols of antibacterial proteins from plant berries respect to those on the wide variety of smaller molecules, obtained usually through ethanol or methanol extraction [25].
In this study, three of simply, fast and common extraction protocols used for proteins were carried out for berries with some modifications [26–29], taking into account both the pH and the presence of strong anionic detergents such as SDS (Figure2A). As far as the protein recovery is concerned, the quantitative comparison among the different extracts showed that the highest yield was obtained with method 3, followed by method 2 and method 1 that gave the lowest protein yield (Figure2B).
However, it is worth noting that the protein quantitation assay on extracts from protocol 3 was affected by the presence of SDS-containing buffer that persisted even after extensive dialysis of the sample, thus interfering with the protein yield results [30].
Figure 2.(A) Representative scheme of the different steps applied for the preparation of crude protein extracts from the yellows berries ofL. europaeus.a: yellow berries;b: pitted berries;c: powdered berries in liquid nitrogen using a mortar and pestle;d: centrifugation of the mixture; e: crude protein extract. Finely ground powder of plant fruit was used as starting material in all three protocols. (B) Table reporting protein yields from berries ofL. europaeususing three extraction protocols. Data are presented as means±standard deviation (s.d.) of three different samples analyzed in triplicate.
The protein pattern of the three extracts was assessed by SDS-PAGE analysis and a representative Coomassie-stained gel is reported in Figure3. The protein extracts 1 and 2 showed a similar electrophoretic profile in contrast to that associated with the extract 3, possibly resulting from the use of SDS in the extraction buffer, which is known to be extremely effective in the solubilization of membrane proteins [31].
Figure 3.SDS-PAGE (10%) analysis of the total protein extracts fromL. europaeusberries using the three methods. Lane 1: molecular weight markers (Thermo Scientific); crude protein extracts obtained by: method 1 (Lane 2); method 2 (Lane 3); and method 3 (Lane 4). Protein bands were detected by Coomassie blue staining. Equal amounts of proteins were loaded for each Lane. The gel is representative of three independent experiments on three different protein preparations.
2.3. Antifungal Activity
An initial in vitro screening was done to evaluate the antifungal activity of all the plant extracts against four of the most common phytopathogenic fungi. As depicted in Figure4, none of the
two extracts at pH 8.0 and 5.0 (Method 1 and 2) showed antifungal activity against any of the test microorganisms, even at the highest amount investigated. In addition, the two protein samples seemed to promote the sporulation ofAspergillus niger(colony diameter of 3.5±0.3 cm and 2.0±0.8 cm for extract 2 and extract 1, respectively after 48 h of incubation), possibly due to the presence in the plant extracts of some additional nutrients which could further stimulate the fungal growth (Figure4C).
As far as the SDS-extract (Method 3) is concerned, it is worth noting that even after extensive dialysis of the samples, a residual amount of detergent persisted in the protein mixtures, resulting in a strong interference with the antifungal and antibacterial activity assays, which require a complete removal of this detergent. For these reasons, the SDS-extracts were not further considered for our investigations.
Figure 4.Antifungal activity assay of plant extract 1 (Method 1) and plant extract 2 (Method 2) against different phytopathogenic fungi: (A)Penicilliumspp; (B)Alternariaspp; (C)Aspergillus niger; (D)Botrytis cinerea. CTRL: each tested fungus without treatment. The plates were incubated at 28◦C for 48 h. The pictures are representative of three independent experiments on three different protein preparations.
2.4. Antibacterial Activity
In order to explore the potential use of the protein samples as antimicrobial agents, the antibacterial activity of the extracts 1 and 2 was evaluated against a panel of bacteria, including 3 strains of Gram-positive(L. monocytogenes,S. aureusMSSA, and MRSA) and 2 strains of Gram-negative bacteria (SalmonellaandE. coli), among those commonly associated with infectious diseases. Specifically, to compare the effect of the two extracts on the growth of the microorganisms under investigation, the MIC and MBC values were determined by using the serial dilution assay. It is known that sodium acetate can affect the bacterial growth [32,33], therefore preliminary experiments were performed in order to assess the effects of different concentrations of acetate on the growth of the foodborne
pathogens, considering that the extract 2 was obtained using acetate as extractant. The obtained results evidenced a linear decrease of bacterial growth rate with the increase in acetate concentration starting from 60 mM (data not shown). For this reason, all the subsequent experiments with extract 2 were performed only after dialysis of the sample in order to have a final concentration of 50 mM acetate that did not interfere with the antimicrobial assays. Interestingly, the tested microorganisms revealed a different sensitivity to the two types of extracts. Overall, the results demonstrated that the extract 1 was less effective in suppressing the microbial growth of all pathogens tested, exhibiting MIC values 2-fold higher than those observed for the acetate-extract. It can be hypothesized that the variation in MIC values between the two plant-fruit samples arose from a diverse nature of the proteins extracted by using the acetate respect to the Tris buffer. Hence, the extract 1 was not considered for any further study based on its weak antibacterial activity. As far as plant fruit extract 2 is concerned (Figure5A), it exhibited an efficient and significant antimicrobial activity againstL. monocytogenes, S. aureusMRSA, andS. Typhimurium, with MIC values ranging from 0.16 to 0.50 mg·mL−1, being S. aureusMRSA the most sensitive bacterial species. Indeed, the protein sample was found to be ineffective againstE. coliandS. aureusMSSA SA4 even at the highest amount (0.50 mg·mL−1) assayed.
To investigate further the antimicrobial effects of the extract 2, the MBC was evaluated revealing that it displayed a strong bactericidal activity againstL. monocytogenesand S. aureus MRSA, with MBC values of 0.38 and 0.20 mg·mL−1, respectively. These results clearly indicated that this protein extract was bacteriostatic at concentrations lower than those required to explain bactericidal activity against L. monocytogenes, being MBC value higher than the corresponding MICs. Instead, the MBC determined againstS. aureusMRSA was on a par with the corresponding MIC (both at about 0.2 mg·mL−1), thus demonstrating that the tested sample should be considered to have a strong bactericidal mode of action.
On the other hand,S.Typhimurium needed protein concentrations higher than 1 mg mL-1to be killed, indicating that the active substances were only bacteriostatic towards this strain. Therefore, according to the results obtained, the Gram-positive bacteria were more sensitive to the plant extract 2 than the Gram-negative microorganisms, presumably as consequence of the different bacterial membrane structures. Specifically, lipopolysaccharides layer and periplasmic space of Gram-negative bacteria could be the reasons of the relative resistance of this class of bacteria to the plant extract 2 treatment.
However, this explanation represents a simplification as other mechanisms could play a role in this process. Interestingly, in relation to the antibacterial spectrum of the crude extract (Figure5A), it is important to emphasize the strong growth inhibition of methicillin-resistanceS. aureusM7 strain (Figure5B,C), which is one of the most pathogenic bacterium resistant to multiple drugs, having acquired resistance to a variety of them.
Antibacterial studies were also performed against a no foodborne Gram-negative pathogen Pseudomonas protegensN, a widespread plant-protecting bacterium isolated from water samples of an irrigation well located in the region of Djebira in Bejaia, northern Algeria [34,35]. The obtained results clearly demonstrated that all the plant extracts under investigation were ineffective to inhibit the growth of the soil microorganism, confirming that theL. europeaus-antibacterial proteins appeared to be less potent both versus pathogenic and not pathogenic Gram-negative bacteria. In accordance with the reported findings concerning the screening of antimicrobial potentiality and taking into account the sensitivity of the tested bacteria, extract 2 andS. aureusMRSA M7 were chosen to perform the further analyses.
Figure 5.(A) Table of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of plant fruit extract 2 against different foodborne pathogens. (B) Antimicrobial test in vitro of plant fruit extract 2 againstS. aureusMRSA M7.CTRL:S. aureusMRSA M7 control; (1) protein extract 2 at 0.08 mg·mL−1concentration; (2) protein extract 2 at 0.16 mg·mL−1concentration (MIC value). (C) MBC value (0.2 mg·mL−1) determined by the standard plate count. Data are presented as means±standard deviation (s.d.) of three different samples analyzed in triplicate. * Significant difference (p<0.05) between the treated and the control samples.
2.5. Spectroscopic Analysis
Many of the colors associated with higher plants are due to the presence of pigment molecules, such as chlorophylls and the carotenoids, which confer them a natural fluorescence. Therefore, the intense color of these pigments makes them ideal candidates for absorption spectroscopy studies, having a unique visible spectrum, which can provide a positive identification [36]. In this context, the pigment content in terms of chlorophyll a, chlorophyll b and carotenoids present in the plant extract 2 was determined by spectrofluorometric analysis, performing their extraction using ethyl acetate as solvent that is considered the best extractant for this class of molecules [37]. As shown in Figure S1, the photosynthetic fluorescence emission spectra obtained from the organic extracts evidenced the presence of three main bands: one of chlorophyll a at 650–684 nm, the second at 642–670 nm, characteristic to chlorophyll b, and the last one at 500–600 nm probably due to carotenoids. The same experiment was performed on the plant extract 2 after dialysis in bags with 10 kDa MWCO (Molecular weight cut-off), revealing that it was completely abolished the fluorescence emission peaks corresponding to the three pigment molecules, which were lost during dialysis (Figure S1). Therefore, it is reasonable to assess that the strong antibacterial activity measured in the extract 2, whose preparation includes dialysis, can be attributed to compounds with a molecular mass higher than 10 kDa.
2.6. Partial Purification of the Active Compounds
With the aim to gain insight into the protein component/s responsible for the antibacterial activity of the extract 2, a partially purification procedure was performed by a combination of ammonium sulphate fractionation and gel filtration chromatography. In the first step, precipitation experiments were conducted subjecting the extract 2 to precipitation using two sequential salt saturation levels (50% and 90%). The pellets resulting from the two precipitation steps were dissolved in 50 mM sodium acetate buffer pH 5.0, extensively dialyzed to remove the ammonium sulphate, tested for antibacterial activity and analyzed by SDS-PAGE (Figure6A).
In vitro antibacterial assessment of the two precipitates (named pellet 50% and pellet 90%) was carried out at the MIC value (0.15 mg·mL−1) determined with the total extract 2 againstS. aureus MRSA (Figure5A) and the results were reported in terms of the change in the Log CFU·mL−1of viable colonies. The bactericidal activity was defined as being equal to 3 Log CFU·mL−1or greater reduction in the viable colony count relative to the initial inoculum [38]. As shown in Figure6B, a rapid reduction in the log of the viable cells counted (−4 Log CFU·mL−1), was detected with both
samples. This acknowledged the fact that the bactericidal activity measured for the extract 2 resulted from the contribution of different protein components. However, given that the total protein yield in the 90% pellet was 5-fold lower than that obtained in 50% sample and taking into account the large amount of the starting material required to allow more detailed investigations, we firstly decided to proceed to the purification of 50% pellet. An important aspect to underline is the complete recovery of the proteins responsible of the antibacterial activity in the plant crude extract after precipitation by ammonium sulphate.
Figure 6.(A) SDS-PAGE analysis of protein fractions. Lane 1: molecular weight markers; Lane 2: plant extract 2; Lane 3: protein sample obtained by 50% ammonium sulphate precipitation; Lane 4: protein sample obtained by 90% ammonium sulphate precipitation. Equal amounts of total proteins were loaded for each lane. The gel is representative of three independent experiments on three different protein preparations. (B) antibacterial effect of pellet 50%, pellet 90% and plant extract 2 samples against S. aureusMRSA M7 reported in terms of change in the Log CFU·mL−1of viable colonies observed between control and treated bacteria at 24 h. Data are representative of three independent experiments on three different protein preparations.
Additional purification step was conducted through the gel filtration chromatography on an SEC-4000 column. The elution profile (Figure7) obtained from 50% pellet, showed five main protein fractions, which were assayed for the antibacterial activity against theS aureusMRSA (Figure8A).
A strong killing activity was exhibited by both protein fractions Fr 1 and Fr 2, with MIC values of 0.01 mg·mL−1and 0.04 mg·mL−1, respectively, which coincided with the MBCs. In contrast, no activity was observed with the remaining protein fractions Fr 3, Fr 4, and Fr 5. Based on the calibration curve of the gel filtration column, Fr 1 and Fr 2 displayed a molecular mass of approximately 600 kDa and 60 kDa, respectively.
On the other hand, the SDS-PAGE analysis of all the gel filtration fractions revealed not only an enrichment of the active compounds (Fr 1 and Fr 2) (Figure8B) but also a possible oligomeric nature of the antibacterial proteins considering the molecular mass determined under native conditions (Figure7).
However, it cannot be excluded that more than one active protein compound could cooperate and contribute to the intrinsic antibacterial activity of theL. europaeusplant fruits.
Figure 7.Elution profile of pellet 50% sample obtained by gel filtration chromatography performed on YARRA™SEC-4000 column in 50 mM sodium acetate buffer pH 5.0 containing 50 mM NaCl. Insert:
Calibration curve of the gel filtration YARRA™SEC-4000 column using protein standards of known molecular masses. The collected fractions are indicated.
Figure 8. (A) Antimicrobial screening assay of gel filtration fractions againstS. aureusMRSA M7.
CTRL:S. aureusMRSA M7 control;Fr 1, Fr 2, Fr 3, Fr 4 and Fr 5: fractions obtained after gel filtration chromatography of the pellet 50% sample. (B) SDS-PAGE analysis of the protein fractions. M: molecular weight markers; Lane 1: pellet 50% sample. Equal amounts of total proteins were loaded for each Lane.
3. Materials and Methods